This invention relates generally to the field of diagnosis and detection of conditions associated with lung injury and pathology.
The lung is a cellularly complex organ that contains more than sixty morphologically distinct types of cells (Stone (1992), Am. J. Respir. Cell Mol. Biol. 6:235-43). These include but are not limited to airway cells (e.g., basal cells, ciliated cells, goblet cells, serous cells, non-ciliated xe2x80x9cClaraxe2x80x9d cells), smooth muscle cells, chondrocytes, endothelial cells (with differing functional properties depending on the type of blood vessel), circulating blood cells, alveolar macrophages, and interstitial cells. The alveolar portion of the lung contains more than 99% of the internal surface area of the lung and is responsible for the essential function of gas exchange between the air and blood compartments. There are two types of alveolar epithelial cells (whose apical surfaces are on the air side of the alveolar surface), called type I and type II cells.
Type I alveolar cells comprise approximately ninety-eight percent of the interstitial surface area of the lung. Type I cells are extremely thin and are primarily responsible for gaseous diffusion between the airspace and the lung capillaries, as well as for the clearance of liquid from the airspaces. The remaining portion of the alveolar surface is primarily composed of type II alveolar cells. Type II cells have several known functions, including production of pulmonary surfactant, production of immunoeffector substances, transport of ions, and repair of the injured alveolus.
Lung injury is often associated with disturbance and destruction of type I alveolar cells; in particular, injury of the alveolar epithelial barrier is thought to be of central importance in the pathogenesis of acute lung injury. For example, type I cells are involved in the earliest changes in the lung in adult respiratory distress syndrome (ARDS). Within a few hours of onset of ARDS the type I alveolar cells swell and become detached from the alveolus"" underlying basement membrane, leaving the gas exchange barrier denuded and prone to leakage of edema fluid. Upon injury to type I cells, type II cells proliferate, spread along the alveolar surface, and eventually differentiate into type I cells.
Methods for early detection and diagnosis of lung injury would greatly benefit patients who have conditions or diseases that are not otherwise detectable using conventional methodology until it is almost too late for successful therapeutic intervention. For example, there are between 30,000 to 100,000 cases annually of ARDS in the U.S. Unfortunately, clinical identification of patients who have developed the syndrome is difficult. Most often, ARDS patients are not identified until the syndrome is full-blown. ARDS is a seriolus, life-threatening condition in which patients remain critically ill in intensive care units for protracted periods of time. The high mortality in ARDS (approx. 50%) has been unchanged from the initial time of the syndrome""s description over twenty years ago.
The presently accepted methods of assessing lung injury require the clinician to obtain biopsy specimens of the lung. The utility of lung biopsy methods in clinical medicine is limited. For example, the invasive nature of the biopsy procedure largely precludes repeated measurements. Interpretation of single biopsies is complicated since lung pathology is often patchy in nature and the size of the biopsy specimen is small. Furthermore, it is difficult to analyze the status of the alveolar epithelial cells obtained at biopsy without time-consuming and costly techniques of electron microscopy. Because electron microscopy is not routinely performed on biopsied lung samples, knowledge about alveolar cells in states of disease is further limited. A non-invasive method for lung injury detection would allow clinicians to make more reliable and accurate diagnoses, monitor the progress of patients, and determine the efficacy of a selected therapeutic regimen, and allow for adjustment and monitoring of therapy.
Unfortunately, the development of diagnostic assays and early therapeutic intervention has been greatly hampered by the lack of human lung cell-specific markers. Specifically, the identification of human type I or type II alveolar cell markers and antibodies that specifically bind such markers has been largely unsuccessful. For example, previously described antibodies that bind to human type I or human type II alveolar cells also bind other alveolar cells and/or bind non-lung tissues (e.g., previously described anti-human type I cell antibodies also bind surface epithelium of distal bronchioles, submucosal glands, skin (superficial keratinocytes), salivary glands, pancreatic acini, breast, prostate, adrenal cortex, and blood vessels (see Singhet al. (1993) Micro. Res. Tech. 26:357-65); previously described anti-human type II cell antibodies also bind to cells found in renal epithelial, Clara cells (airway cells), and cells of other organs (see Singh et al. (1985) Clin Exp. Immunol. 60:5791185; Kotas et al. (1991) Ped. Pulmunol. 10:260-6; Sahali et al. (1993) Am. J. Pathol. 142:1654-67)). Due to the cross-reactivity of such previously described antibodies, these antibodies have only limited uses in, for example, in vitro and in vivo specific and sensitive diagnostic methods for lung disease. Previously identified markers for rodent type I alveolar cells (Dobbs et al. (1988) Biochimica Biophysica Acta 970:146-56) do not cross-react with human cells as determined by morphological and biochemical criteria, making them useless in human applications.
Biochemical and molecular markers for other tissues have proven invaluable, in both diagnosis and management of human disease. For example, the biochemical and molecular markers available for cardiac injury (e.g., acute myocardial infraction and ischemia) have been used in cardiac disease to identify when cardiac injury has occurred, to quantitate the extent of injury, and to determine whether there is ongoing injury. Quantitation of injury hasalso been extremely important in evaluating the therapeutic strategies to treat injury. The potential value of human lung cell markers has been validated in various models of rodent lung injury, in which the airspace liquid content of RT140, a rat type I cell integral membrane protein, correlated directly with the extent of epithelial injury assess by morphologic criteria, ((1995) Am. J. Physiol. 268:L181-6; (1997) Am. J. Physiol. 272:L631-8). The lack of analogous markers for human lung injury has greatly impaired the clinician""s ability to determine whether lung injury exists, what the prognosis might be, or to determine whether specific therapeutic intervention is of benefit.
The ability to identify patients having lung injury, detect injury at the time of its development, and determine the severity of injury would greatly aid in the early detection of conditions associated with lung injury such as ARDS, and the treatment of such patients before it is may be too late for the patient to benefit. Moreover, a non-invasive method to detect lung injury would provide a more reliable and accurate diagnosis, and allow clinicians to follow a patient""s progress and adjust therapy accordingly. Therefore, there is a distinct need in the field for markers for lung injury, especially for acute lung injury, and for methods to readily detect such markers. The present invention addresses this problem.
The invention features monoclonal antibodies specific for human type I alveolar cells or for human type II alveolar cells. The invention also features methods of detecting lung injury in a subject using these monoclonal antibodies.
Accordingly, in one aspect the invention features methods for detection of lung injury in a human subject, the method comprising the steps of: a) contacting a specific anti-human type I or type II alveolar cell monoclonal antibody (MAb) with a test sample from a subject suspected of having lung injury, wherein the sample is suspected of comprising a human type I or type II alveolar cell-specific polypeptide; and b) detecting binding of the monoclonal antibody to the human type I or type II alveolar cell-specific polypeptide in the sample, where detection of binding of the MAb to the human type I or type II alveolar cell-specific polypeptide in the sample at a level substantially altered relative to a level in a normal subject is indicative of lung injury in the subject. In a preferred embodiment, the polypeptide for human type I or type II alveolar cells may be either HTI56 or HTII280, respectively. In preferred embodiments, the test sample is an edema fluid sample, a plasma sample, or a urine sample.
In an especially preferred embodiment, detection of MAb binding to the type I or type II alveolar cell-specific polypeptide is accomplished by immobilizing polypeptides in the test sample on a solid support; contacting the immobilized polypeptides with the anti-humnan type II alveolar cell-specific monoclonal antibodies, wherein the antibody is detectably labeled; and detecting binding of the specific monoclonal antibody using a luminometer.
In other aspects, the invention features human type I and type II cell-specific MAbs. The MAbs of the invention can be produced by immunizing a non-human mammal with isolated human type I or type II alveolar cells; producing a hybridoma cell from spleen cells of the immunized mammal; and identifying a hybridoma cell producing an anti-human type I or type II alveolar cell antibody by specific binding of hybridoma supernatant to human type I or type II alveolar cells in a cytospin preparation or tissue, where the anti-type I or type II alveolar cell MAbs produced are characterized by specific binding to type I or type II alveolar cells, respectively, specific binding to lung tissue, and substantially no detectable binding to tissue homogenates of brain, skin, trachea, lung, esophagus, stomach, intestine, urinary bladder, blood, kidney, or spleen as detected by Western blot. In preferred embodiments, the anti-type I cell MAb binds a polypeptide of about 55-56 kDa (e.g., HTI56); the anti-type II cell MAb binds a polypeptide of about 240-280 kDa (e.g, HTII280).
The invention also features kits useful in detection of lung injury in a subject, the kit comprising a MAb specific for human type I alveolar cells or for human type II alveolar cells.
The invention further features isolated HTI56 and HTII280 polypeptides.
A primary object of the invention is to provide a non-invasive method (e.g., without lung biopsy) for detecting lung injury, which in turn allows for diagnosis of a variety of conditions or diseases associated with such lung injury.
One advantage of the invention is that it provides specific markers for human type I and type II alveolar cells, which markers can be used in a variety clinical situations such as diagnosis of the extent of lung injury due to various processes (e.g., ARDS, infection, inflammatory lung disease), assessment of the state of maturity of fetal lungs, targeting of drugs for delivery to specific cells or to specific regions of the lung, and treatment of diseases such a Pneumocystis carinii, in which the pathogen attaches to alveolar cells.
Another advantage is that lung injury detection method of the invention can be used with biological samples such as lung fluid edema, blood and blood-derived samples and urine, which do not involve invasive techniques (e.g., lung tissue biopsy).
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the methodology and compositions as more fully set forth below.